Bottom Line:
We also identified a novel physical interaction in Schwann cells, between Mtmr2 and discs large 1 (Dlg1)/synapse-associated protein 97, a scaffolding molecule that is enriched at the node/paranode region.Dlg1 homologues have been located in several types of cellular junctions and play roles in cell polarity and membrane addition.We propose that Schwann cell-autonomous loss of Mtmr2-Dlg1 interaction dysregulates membrane homeostasis in the paranodal region, thereby producing outfolding and recurrent loops of myelin.

ABSTRACTMutations in MTMR2, the myotubularin-related 2 gene, cause autosomal recessive Charcot-Marie-Tooth (CMT) type 4B1, a demyelinating neuropathy with myelin outfolding and azoospermia. MTMR2 encodes a ubiquitously expressed phosphatase whose preferred substrate is phosphatidylinositol (3,5)-biphosphate, a regulator of membrane homeostasis and vesicle transport. We generated Mtmr2- mice, which develop progressive neuropathy characterized by myelin outfolding and recurrent loops, predominantly at paranodal myelin, and depletion of spermatids and spermatocytes from the seminiferous epithelium, which leads to azoospermia. Disruption of Mtmr2 in Schwann cells reproduces the myelin abnormalities. We also identified a novel physical interaction in Schwann cells, between Mtmr2 and discs large 1 (Dlg1)/synapse-associated protein 97, a scaffolding molecule that is enriched at the node/paranode region. Dlg1 homologues have been located in several types of cellular junctions and play roles in cell polarity and membrane addition. We propose that Schwann cell-autonomous loss of Mtmr2-Dlg1 interaction dysregulates membrane homeostasis in the paranodal region, thereby producing outfolding and recurrent loops of myelin.

fig3: Myelin outfoldings in Mtmr2- peripheral nerves. (A–D) Serial transverse sections of Mtmr2- sciatic nerves at 4 wk old. Arrows indicate a fiber with myelin outfoldings extending from the node (A) throughout the internode (B–D). (E and F) Transverse sections of mutant sciatic nerves at 7 wk (E) and 6 mo old (F), where arrows indicate myelin outfoldings; open arrow (E) indicates a myelin infolding, and the arrowhead indicates a comma-shaped myelin outfolding. (G–I) Longitudinal sections of sciatic nerves from Mtmr2- mice, where arrows indicate the onset of myelin outfoldings and recurrent loops at the paranodes/juxtaparanodes. The asterisk marks a loop of Schwann cell membrane protruding into the axonal space (infolding). (J–M) Cross sections of saphenous nerves from a wild-type (J) and a mutant (L) mouse, or quadriceps nerves from a wild-type (K) and a (M) mutant mouse. Myelin outfoldings are evident (L and M, arrows) in both mutant nerves; the arrowhead indicates a comma-shaped myelin outfolding. (N and O) Cross sections of digital nerves from a wild-type (N) or a mutant (O) mouse, where myelin outfoldings are present in almost all myelinated fibers. Bar: (A–F and J–O) 15 μm; (G–I) 5 μm.

Mentions:
Necropsy of Mtmr2- mice revealed normally formed organs without obvious abnormality. Histology of the brain, spinal cord, and muscle (including ATPase isotype staining) was grossly normal at 2 mo old. Only peripheral nerves demonstrated abnormalities (Fig. 3 and not depicted). The predominant changes were seen in myelin sheaths; transverse semithin sections revealed myelin outfolding that appeared as “comma”-shaped extensions of both myelin and axoplasm (Figs. 3 E and 4 A), or recurrent loops that appeared as one to five satellite myelinated axons around a larger myelinated axon (Fig. 3, E and M; and Fig. 4, B and C). Less frequently, myelin protruded into the axon, producing a myelinated fiber within a larger myelinated axon (Figs. 3 E and 4 B). The number of fibers containing myelin outfoldings and loops increased progressively in sciatic nerves of mice examined at 1 mo (4.1%), 2 mo (5.2%), 4 mo (7.7%), and 6 mo old (11%; Fig. 3, compare E with F). The complexity of myelin outfoldings also progressed with age, because the number of fibers showing three or more satellite loops increased from 2.5% at 1 mo to 6.25% at 2 mo, 10% at 4 mo, and 8.4% at 6 mo old. Almost all large myelinated fibers in digital nerves of mice contained myelin outfoldings (Fig. 3 O), suggesting that the morphological alterations were length dependent.

fig3: Myelin outfoldings in Mtmr2- peripheral nerves. (A–D) Serial transverse sections of Mtmr2- sciatic nerves at 4 wk old. Arrows indicate a fiber with myelin outfoldings extending from the node (A) throughout the internode (B–D). (E and F) Transverse sections of mutant sciatic nerves at 7 wk (E) and 6 mo old (F), where arrows indicate myelin outfoldings; open arrow (E) indicates a myelin infolding, and the arrowhead indicates a comma-shaped myelin outfolding. (G–I) Longitudinal sections of sciatic nerves from Mtmr2- mice, where arrows indicate the onset of myelin outfoldings and recurrent loops at the paranodes/juxtaparanodes. The asterisk marks a loop of Schwann cell membrane protruding into the axonal space (infolding). (J–M) Cross sections of saphenous nerves from a wild-type (J) and a mutant (L) mouse, or quadriceps nerves from a wild-type (K) and a (M) mutant mouse. Myelin outfoldings are evident (L and M, arrows) in both mutant nerves; the arrowhead indicates a comma-shaped myelin outfolding. (N and O) Cross sections of digital nerves from a wild-type (N) or a mutant (O) mouse, where myelin outfoldings are present in almost all myelinated fibers. Bar: (A–F and J–O) 15 μm; (G–I) 5 μm.

Mentions:
Necropsy of Mtmr2- mice revealed normally formed organs without obvious abnormality. Histology of the brain, spinal cord, and muscle (including ATPase isotype staining) was grossly normal at 2 mo old. Only peripheral nerves demonstrated abnormalities (Fig. 3 and not depicted). The predominant changes were seen in myelin sheaths; transverse semithin sections revealed myelin outfolding that appeared as “comma”-shaped extensions of both myelin and axoplasm (Figs. 3 E and 4 A), or recurrent loops that appeared as one to five satellite myelinated axons around a larger myelinated axon (Fig. 3, E and M; and Fig. 4, B and C). Less frequently, myelin protruded into the axon, producing a myelinated fiber within a larger myelinated axon (Figs. 3 E and 4 B). The number of fibers containing myelin outfoldings and loops increased progressively in sciatic nerves of mice examined at 1 mo (4.1%), 2 mo (5.2%), 4 mo (7.7%), and 6 mo old (11%; Fig. 3, compare E with F). The complexity of myelin outfoldings also progressed with age, because the number of fibers showing three or more satellite loops increased from 2.5% at 1 mo to 6.25% at 2 mo, 10% at 4 mo, and 8.4% at 6 mo old. Almost all large myelinated fibers in digital nerves of mice contained myelin outfoldings (Fig. 3 O), suggesting that the morphological alterations were length dependent.

Bottom Line:
We also identified a novel physical interaction in Schwann cells, between Mtmr2 and discs large 1 (Dlg1)/synapse-associated protein 97, a scaffolding molecule that is enriched at the node/paranode region.Dlg1 homologues have been located in several types of cellular junctions and play roles in cell polarity and membrane addition.We propose that Schwann cell-autonomous loss of Mtmr2-Dlg1 interaction dysregulates membrane homeostasis in the paranodal region, thereby producing outfolding and recurrent loops of myelin.

ABSTRACTMutations in MTMR2, the myotubularin-related 2 gene, cause autosomal recessive Charcot-Marie-Tooth (CMT) type 4B1, a demyelinating neuropathy with myelin outfolding and azoospermia. MTMR2 encodes a ubiquitously expressed phosphatase whose preferred substrate is phosphatidylinositol (3,5)-biphosphate, a regulator of membrane homeostasis and vesicle transport. We generated Mtmr2- mice, which develop progressive neuropathy characterized by myelin outfolding and recurrent loops, predominantly at paranodal myelin, and depletion of spermatids and spermatocytes from the seminiferous epithelium, which leads to azoospermia. Disruption of Mtmr2 in Schwann cells reproduces the myelin abnormalities. We also identified a novel physical interaction in Schwann cells, between Mtmr2 and discs large 1 (Dlg1)/synapse-associated protein 97, a scaffolding molecule that is enriched at the node/paranode region. Dlg1 homologues have been located in several types of cellular junctions and play roles in cell polarity and membrane addition. We propose that Schwann cell-autonomous loss of Mtmr2-Dlg1 interaction dysregulates membrane homeostasis in the paranodal region, thereby producing outfolding and recurrent loops of myelin.